TNF ligand-receptor interactions serve diverse functions in immune system regulation. Fas, a member of the TNF receptor family is an important regulator of lymphocyte homeostasis and can cause systemic autoimmunity when genetically mutated. Althuogh a number of signaling proteins mediating TNF receptor signals have been identified, the cell biology of receptor signaling: where signaling events take place within the cell and whether subcellular localization plays a role in regulating TNF receptor signaling, remains relatively unknown. Understanding of this process is important for devising better ways to modulate signaling through these receptors for therapeutic goals. In previous work, we established that TNF receptors must pre-associate into mutimeric complexes to signal efficiently. Preassociation is mediated by an N-terminal pre-ligand association domain (PLAD) For the apoptosis-inducing receptor Fas (CD95, APO-1), we have more recently characterized much larger microscopically visible surface receptor clusters initiated by antibody or ligand that are dependent on the death domain and are required for caspase-8 activation and apoptosis signaling. Additionally, we have found a role for lipid raft microdomains in regulating the effciciency of Fas signaling. With Dr. Lenardo, we are investigating impairments in the Fas receptor clustering process in cells from human patients harboring various mutations in Fas and downstream signaling proteins. With Dr. Subramanian, we are studying the ultrastructural features of receptor clusters with the goal of 3-D reconstruction of the signaling complex at the plasma membrane. We are also investigating whether unliganded receptors can be drawn into these clusters through lateral association of signaling adapters. We have defined a region of the Death Effector Domain of the Fas-associated adapter protein FADD that mediates self-association and is essential for receptor signaling. In collaboration with Dr. Screaton we have produced recombinant protein encoding the preassociation domain in the extracellular portion of Fas/CD95 in a form that blocks ligand-induced apoptosis. With Drs. Hull and Kastner, we are developing a system to study signaling abnormalities in cells expressing mixtures of wild- type and mutant TNF-R1 receptors from patients with inherited mutations in this receptors and a periodic fever syndrome (TRAPS).